There has been increasing interest and progress in the field of membrane protein research over the last years. The high interest in membrane proteins is due to the fact that they play an important role in several biological processes such as ion transport, recognition of molecules, signal transduction, etc. G-protein coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play an important part in signal transduction by converting extracellular stimuli including light, smells, neurotransmitters and hormones, into intracellular signals. Currently, more than 30% of marketed medicines act on GPCRs, which are considered as attractive targets for new medicines. Structure-based drug discovery and high throughput screening (HTS) for novel compounds active on a receptor of interest have now become an integrated technology in pharmaceutical laboratories.
Although membrane proteins represent 20% to 30% of all genes in prokaryotes as well as in eukaryotes, only little is known about structure and function relationship of membrane proteins. This can be largely attributed to the low natural expression of membrane proteins, to their hydrophobic character, which complicates overexpression of functional membrane proteins, as well as to difficulties during their purification and crystallization. By way of example, only for seven GPCRs high-resolution structures have been characterized: rhodopsin, the β1 and β2 adrenergic receptors, the adenosine 2A receptor, and more recently the CXCR4 receptor, the dopamine D3 receptor and the histamine H1 receptor. Whereas rhodopsin was purified and subsequently crystallized from unmodified protein isolated from native tissue (a lone exception to the rule of low expression levels), these other GPCRs required expression in recombinant systems, stabilization of an inactive state by an inverse agonist/antagonist and biochemical modifications to stabilize the receptor protein (e.g., Rasmussen et al., 2007, Nature 450:383; Rosenbaum et al., 2007, Science, 318:1266; Warne et al., 2008, Nature 454:486; Wu et al., 2010, Science 330:1066; Chien et al., 2010, Science 330:091; Shimamura et al., 2011, Nature 475: 65. Up till now, most unravelled structures have the third cytoplasmic loop replaced for the very stable bacteriophage T4 lysozyme. This fusion protein might however not represent the true natural conformation of the GPCR. Therefore, the structures need to be analyzed with great care when performing ligand screening and drug design. Besides that, evidence from functional and biophysical studies shows that GPCRs can exist in multiple functionally distinct conformational states (Kobilka and Deupi 2007, Trends Pharmacol Sciences 28:397). While this structural plasticity and dynamic behavior is essential for normal function, it contributes to their biochemical instability and difficulty in obtaining high-resolution crystal structures. Only recently it became possible to obtain structures of an active state of a GPCR, making use of stabilizing Nanobodies (Rasmussen et al., 2011, Nature 469: 175).
Most membrane proteins express at low levels in non-engineered eukaryotic cells. Eukaryotic membrane proteins have been successfully overexpressed in bacteria, yeast, mammalian cell lines and insect cells (reviewed in Freigassner et al., 2009, Microb Cell Fact. 8:69). However, expression levels are still rather low and for the majority of these receptors a 5- to 10-fold increase in expression level would lead to sufficient material for subsequent experiments, especially protein purification and characterization, structural and pharmacological studies. For example, expression of eukaryotic membrane proteins in prokaryotic systems mostly leads to poor expression levels. Besides, in many cases the protein ends up in denatured form in inclusion bodies and the necessity of complicated refolding processes has hampered the success. Expression in yeast is a valuable alternative for the expression of eukaryotic membrane proteins. Yeast cells are easy to handle, and can grow in fermentors to very high cell densities. Different techniques to increase the expression levels of the membrane protein have been used in yeast such as lowering the induction temperature, adding antagonist, DMSO or histidine to the induction medium (André et al., 2006, Protein Sci. 15:1115). Other approaches have been taken to enhance membrane protein surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones (reviewed in Dunham and Hall, 2009, Trends Biotechnol. 27:541). It remains a challenge, however, to significantly improve total yield, conformational stability and/or functionality of wild-type surface expressed membrane protein.
Thus, it would be advantageous to have alternative expression systems that permit higher heterologous expression of native membrane proteins in a particular conformation. This would greatly facilitate the whole trajectory of drug discovery efforts on membrane proteins as therapeutic targets.